DISEASES OF AQUATIC ORGANISMSDis Aquat Org

Vol. 75: 51–59, 2007 Published March 29

INTRODUCTION

Members of the strigeid digenean genus Diplosto-mum Nordmann, 1832 and the closely related genusTylodelphys Diesing, 1850 are important parasiticpathogens of farmed and wild stocks of fish, and infec-tions are known to induce blindness, emaciation, cra-nial deformation, poor growth and, in some species,host mortality (Shariff et al. 1980, Chappell 1995,Pylkkö et al. 2006).

A parasite’s affinity or specificity for particular tis-sue/organs sites may result from a number of factors

that may be driven by the specific nutritional and/orenvironmental requirements of the parasite (Crompton1976), the consequences of inter-specific competitionfor resources and subsequent niche separation be-tween species (Holmes & Price 1985), or by attempts toevade the host’s chemical and cellular immunedefences (Price 1987, Sukhdeho & Mettrick 1987,Sukhdeho & Sukhdeho 1994).

The current study on a population of Diplostomumphoxini (Faust, 1989)-infected Phoxinus phoxinus (L.)in the River Endrick, Scotland, found metacercariaeprincipally within the cerebellum, the medulla oblon-

© Inter-Research 2007 · www.int-res.com*Email: dzb@unife.it

Histopathological and ultrastructural observationsof metacercarial infections of Diplostomum phoxini

(Digenea) in the brain of minnowsPhoxinus phoxinus

Bahram Sayyaf Dezfuli1,*, Silvia Capuano1, Edi Simoni1, Luisa Giari 1, Andrew Paul Shinn2

1Department of Biology, University of Ferrara, Via Borsari, 46, 44100 Ferrara, Italy2Institute of Aquaculture, University of Stirling, Stirling FK9 4LA, UK

ABSTRACT: The spatial distribution and histopathological changes induced by metacercariae of thedigenean trematode Diplostomum phoxini (Faust, 1918) in the brains of European minnows Phoxinusphoxinus (L.) from the River Endrick, Scotland, were studied by light and electron microscopy. Post-mortem examination of a sample of 34 minnows revealed that 50% (n = 17) of the population wasinfected with 13.7 ± 2.6 (mean ± SE; range 1 to 38) metacercariae per infected host. Serial histologi-cal sections of the infected minnow brains revealed that the metacercariae were unevenly distributedthroughout the brain, with aggregations occurring in the cerebellum, the medulla oblongata and theoptic lobes. In fish with highest intensities of infection, over 40% of the cerebellar area and about30% of the medulla oblongata area were occupied by larvae. Metacercariae disrupt the integrity ofbrain tissue, with individuals being found in small pockets surrounded by cellular debris. Metacer-cariae were rarely encountered on the surface of the brain. Electron microscopic examination ofinfection sites revealed that the granular layer surrounding metacercariae was necrotic, exhibitednuclear degradation and was marked by vacuolation of the cytoplasm. Rodlet cells, the only inflam-matory cell types recorded in this study, were found only in parasitized brains and in close proximityto the teguments of metacercariae. It is hypothesised that secretions released from the teguments ofmetacercariae are a counter response to protect the metacercariae from the fish brain’s cellulardefence mechanisms.

KEY WORDS: Brain infection · Digenean trematode · Phoxinus phoxinus · Rodlet cells ·Histopathology · Ultrastructure

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Dis Aquat Org 75: 51–59, 2007

gata and the optic lobes, centres of the brain that areresponsible for host motor activity, sensory functionsand vision (Barber et al. 2000, Shirakashi & Goater2005). Until recently, metacercariae were generallyregarded as immature, non-feeding and metabolicallyinactive stages waiting for transmission to the next/definitive host (Bush et al. 2001, Poulin & Latham2003). However, a recent study by Goater et al. (2005)demonstrated via experimental infection that themetacercariae of Ornithodiplostomum ptychocheilusFaust, 1917 in Pimephales promelas Rafinesque, 1820undergo a series of complex developmental changesassociated with their feeding on host brain tissue.

A histological examination of Phoxinus phoxinusheads in the current study revealed the presence ofrodlet cells only in infected brains, notably in theepithelium lining the ventricles of the optic lobes. It isbelieved that rodlet cells play a role within the piscineinflammatory response (Dezfuli et al. 2000, Manera &Dezfuli 2004, Reite 2005, Reite & Evensen 2006). Wefound rodlet cells only within the infected brain of afish, and our findings are briefly compared with theinformation available on their presence in other fish-parasite systems (Dezfuli et al. 1998, 2000, 2002, 2003,Reite 1998, Bosi et al. 2005). While there have beennumerous ultrastructural studies on metacercariae(Stein & Lumsden 1971, Strong & Cable 1972, So &Wittrock 1982, Faliex & Biagianti 1987, Wittrock et al.1991, Goater et al. 2005), most focused on the develop-ment of the larval epithelial layer within the intermedi-ate host, and only a few of these studies focused on theinteractions between host tissues and the parasite(Baghaei 1981, So & Wittrock 1982, Faliex & Biagianti1987, Wittrock et al. 1991). Fewer again used transmis-sion electron microscopy (TEM) to examine the cysts ofstrigeoid digeneans (Bibby & Rees 1971, Mitchell 1974,Baghaei 1981, Goater et al. 2005).

The present study provides information on (1) theoccurrence of Diplostomum phoxini metacercariae inthe brains of minnows Phoxinus phoxinus, with partic-ular comments on the interface region between thehost and the parasite; (2) light and electron micro-scopic observations on histopathological changes inbrain tissue; (3) the location of metacercariae, the dam-age they cause in key regions of the brain and theireffect on host vision and motor activity; and (4) therelationship between brain cells, inflammatory cellsand D. phoxini metacercariae.

MATERIALS AND METHODS

Thirty-four minnows Phonixus phoxinus, were hand-netted from the River Endrick, Stirlingshire, Scotland(56° 03’ N, 4° 27’ W) and transferred to the laboratory in

river water where they were maintained in aeratedaquaria until being processed. Fish were subsequentlysacrificed by exposure to a lethal dose of the anaes-thetic MS-222 (Sandoz), and were then weighed (meanweight ± SE: 0.21 ± 0.01 g), measured (mean totallength ± SE: 30.57 ± 0.55 mm) and processed for histol-ogy. After severing the spinal cord, 28 heads wereremoved and fixed in chilled (4°C) Bouin’s fluid for 7 hbefore being transferred into chilled 70% alcohol forstorage until being processed for paraffin embeddingby routine methods using an automatic tissue proces-sor (Shandon Citadel 2000 Tissue Processor, ShandonSouthern Products). Histological sections (5 µm thick)were cut and stained with a haematoxylin and eosin(H&E), Azan-Mallory, periodic acid Schiff (PAS) or analcian blue/PAS stain. Light photomicrographs weretaken using a Nikon microscope Eclipse 80i. Thedimensions of Diplostomum phoxini key features in theacquired images were subsequently determined usingcomputerised image analysis software (Lucia G 4.8,Laboratory Imaging). For the TEM study, 6 P. phoxinusheads were fixed for 2 h in a chilled (4°C) 2% glu-taraldehyde solution buffered at pH 7.2 with 0.1 Msodium cacodylate, and were then rinsed in 0.1 Msodium cacodylate buffer containing 6% sucrose forapproximately 12 h. The minnow heads were thenpost-fixed in 1% osmium tetroxide in the same bufferfor 2 h, dehydrated through a graded ethanol series,transferred to propylene oxide and then embedded inan Epoxy-Araldite mixture. Semi-thin sections (1.5 µm)were cut on a Reichert Om U2 ultramicrotome usingglass knives and stained with toluidine blue. Ultra-thinsections (90 nm) were contrasted in a 50% alcohol-uranyl acetate solution and lead citrate, and examinedusing a Hitachi H-800 electron microscope operated at80 kV.

RESULTS

Post-mortem histological examination of 34 minnowheads revealed that 50% (n = 17) were infected with13.7 ± 2.6 (mean ± SE; range 1 to 38) Diplostomumphoxini metacercariae per infected fish. Measurement(mean ± SE) of the metacercariae (239.75 ± 11.51 µmlong by 103.05 ± 5.88 µm wide) indicated that theinfections were well established (as opposed to beingcharacterised by diplostomules that had recentlyentered the brain). Fig. 1 & 2 shows uninfected and D.phoxini-infected brains. The most frequently occupiedregions of the brain included the medulla oblongata(cortex and centre, Fig. 3), the optic lobes, the cerebel-lum and its superior lobe, and the cavity between thesuperior lobe and the anterior part of the medullaoblongata. In addition to these areas, 3 fish were also

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Dezfuli et al.: Metacercariae of minnow brain

found to have metacercariae within the superior lobeof the cerebellum. However, the inferior lobe of thecerebellum, the pituitary, the olfactory lobes and theolfactory bulbs were largely free of metacercariae. Inbrains with more than 15 larvae, over 40% of the cere-bellar area and about 30% of the medulla oblongata

area were occupied by metacercariae; consequentially,a low proportion of the nervous tissue was found toremain within these regions (Figs. 2 & 4). Meta-cercariae within the medulla oblongata and the cere-bellum were found within large cleared areas sur-rounded by brain cells and cellular debris.

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Figs. 1 & 2. Fig. 1. Phoxinus phoxinus. Sagittal section through an uninfected brain of P. phoxinus. Note the integrity of eachregion of the brain. OLFL: olfactory lobes; OPL: optic lobe; SCL: superior cerebellar lobe; CC: cerebellar cavity; ICL: inferior cere-bellar lobe; MO: medulla oblongata. Short arrow shows the direction of the spinal cord. Scale bar = 250 µm. Fig. 2. Sagittal sec-tion through the entire head of a P. phoxinus with a heavy infection of Diplostomum phoxini metacercariae. A high proportion ofthe medulla oblongata and regions of the cerebellum have disappeared and are occupied by numerous metacercariae (dashed

circle). OLFB: olfactory bulbs; SC: spinal cord. Scale bar = 500 µm

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Figs. 3 to 9. Phoxinus phoxinus. Different regions of Diplostomum phoxinus-infected brains. Fig. 3. D. phoxini metacercariae canbe seen in the centre (arrows) and on the periphery (arrowheads) of the medulla oblongata, and are sometimes surrounded by aclear area. Scale bar = 250 µm. Fig. 4. In a heavily D. phoxini-infected brain, only a small proportion of the cerebellum (SCL) andmedulla oblongata (MO) regions were found intact. Scale bar = 250 µm. Fig. 5. Attachment of 2 metacercariae to host brain tis-sue, with detached pieces of host nervous tissue within their ventral/oral suckers (arrows). Scale bar = 30 µm. Fig. 6. A high mag-nification of the ventral sucker on a metacercaria. Note the presence of nervous elements within the sucker (arrow). Scale bar =10 µm. Fig. 7. Metacercaria encystment within the optic lobe. Host cells (arrowheads) are attached to the larval tegument; arrowsshow granule cells of optic lobe. Scale bar = 30 µm. Fig. 8. Two rodlet cells (arrowheads) within the granule cells (arrow)surrounding a metacercaria (�). Scale bar = 10 µm. Fig. 9. Rodlet cells (arrowheads) in the surface of the ventricle of the optic

lobe. One rodlet cell (arrow) is in the process of discharging its contents. Scale bar = 10 µm

Dezfuli et al.: Metacercariae of minnow brain

Parasites were commonly observed attached to thehost tissue by their ventral and/or oral suckers, or wereobserved with detached pieces of brain tissue withinthem (Figs. 5 & 6). Single metacercaria, in 2 separatefish, were also noted embedded within the tissues of anolfactory bulb (Fig. 7). Although infections in the opticlobes were observed to be lower than those seen in thecerebellum or the medulla oblongata, metacercariaefound here and within the medulla oblongata (Fig. 7)were observed to be in intimate contact with granulecells of the brain. Granule cells, which are typicallydark in appearance and possess circular-shaped nucleiand a diffuse cytoplasm, form the granular layer of theoptic lobe ventricles (Fig. 7).

The presence of rodlet cells scattered among thegranule cells of the optic lobes of infected fish only(Figs. 8 & 12) is of particular interest. In each case, therodlet cells were observed on the surface of the ventri-cles of the optic lobes and in close proximity to ametacercaria, where some rodlet cells were seen to bedischarging their contents (Fig. 9). Rodlet cells werealso commonly encountered within the periphery ofthe cerebellum in all Diplostomum phoxini-infectedbrains. The rodlet cells, which appeared to be large indimension and measured 12.12 ± 0.19 µm (major axis)by 8.59 ± 0.16 µm (minor axis), typically contained ahigh number of club-shaped structures (rodlets) thatpossess a crystalline core and a vesicular, foam-likecytoplasm that did not contain any other evidentorganelles (Fig. 12).

Thirteen of the 28 minnows heads prepared for waxembedding and 4 of the 6 heads prepared for the TEMstudy were infected with metacercariae: 1 to 38 parasitesper infected head were observed in the former study,and 1 to 3 in the latter. From these samples, only metac-ercariae within the optic and olfactory lobes wereobserved to make intimate contact with host tissue. ATEM study of these regions suggested that the regionwhere the parasite interfaces with the host can be bro-ken down into 2 zones. The inner zone, which is adjacentto the metacercaria, possesses a large number of whitevacuoles (Fig. 10). In histological sections prepared forlight microscopy, this zone appears as large white spacessurrounding metacercariae (Figs. 3 & 4). Within the op-tic lobe, the inner zone of granule cells, which are inclose proximity to a metacercaria, are notably flattenedand in a degenerative state typified by severe vacuola-tion and organelle regression (Figs. 10 & 11). The nucleiof the cells in this region also appear necrotic, showkaryolysis, chromatin margination and condensation(Fig. 11), and in some cases nuclear swelling (Fig. 11).

By contrast, the outer zone of the host-parasite inter-face appears more compact in density and containsseveral granule cells in a state of degeneration. Theultrastructural changes observed in the olfactory lobe

were similar to those observed for the optic lobe, withcells in a degenerative state that decreased in severitywith distance from the metacercariae (Fig. 13a). Thecharacteristics of the granule cells that are located at adistance from metacercariae and show few histopatho-logical changes (Fig. 13a) are similar to the granulecells observed in the brains of uninfected fish(Fig. 13b). The only observed difference betweenthese 2 groups of cells was that the granule cells in theuninfected fish brains contained more glycogen.

Close examination of the host-parasite interactionzone also revealed the occurrence of a high number ofsecretory vesicles (Fig. 14). These vesicles, which arearranged regularly within the tegument of the metac-ercariae, open into the interfacing zone, where theycan be seen releasing their cellular contents, whichconsist of a loose microfilamentous material (Fig. 14).In many instances, the secreted material was observedin contact with the host brain cells, which appeared tobe in a partial or total degenerative state (Fig. 14).

DISCUSSION

The current study found that the sub-sample ofPhoxinus phoxinus collected from the River Endrickpopulation were infected by Diplostomum phoxini thatfavor the cerebellum, the medulla oblongata and theoptic lobe regions of the brain. Metacercariae coloniz-ing these sites are suitably placed to alter the sensoryand motor functions of the host. For example, the cere-bellum of fish is believed to be linked to a variety ofcognitive functions (Rodriguez et al. 2005), and itsremoval from a dogfish Mustelus canis Mitchillresulted in a marked reduction in motor and sensoryperformance (Karamyan 1956). The medulla oblongatais known to control a range of involuntary responsessuch as breathing, digestion and heart rate (Bernstein1970), while the optic lobes are the primary vision-processing centre in teleost fishes (Guthrie 1986);herein, visual information regarding movement, colorand shape are analyzed, and stimuli from the lateralline system are received (Springer et al. 1977,Kortrschal et al. 1991).

An investigation by Barber & Crompton (1997) thatexamined the distribution of Diplostomum phoxini inthe brains of 2 populations of minnows found that the 2populations favoured particular regions of the brainand almost totally neglected others. The finding fromBarber & Crompton’s (1997) study suggests that indi-vidual D. phoxini or populations of D. phoxini mayfavour certain sites over others, which—once dam-aged—facilitates their transmission to the final host bycompromising the anti-predatory behaviour and per-formance of their fish host.

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Almost all the Diplostomum phoxini metacercariaeencountered in the current study were fully developed,i.e. were not migrating diplostomules that had justentered the brain. Bush et al. (2001) and Poulin &Latham (2003) assumed that, developmentally andecologically, this part of the digenean life-cycle is a

resting stage. However, this suggestion was mostlikely based on ultrastructural and cytochemical obser-vations made on the cyst wall of fully developedmetacercariae. Based on ultrastructural examinationsof the metacercarial tegument, it was suggested thatthe opportunities for absorption across the tegument

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Figs 10 to 14. Phoxinus phoxinus. Electron micrographs of uninfected and Diplostomum phoxini-infected brains. Fig. 10. Highmagnification of the host-parasite interface zone. Numerous white vacuoles (arrowheads) close to the metacercaria tegument (�)are evident, as are a number of flattened, degenerating nuclei (arrows). A number of secreting bodies within the tegument of themetacercaria are also visible (white arrows). Scale bar = 5 µm. Fig. 11. High magnification of the region between D. phoxini andP. phoxinus. Micrograph shows a large number of necrotic nuclei (arrowheads) with karyolysis, chromatin margination andcondensation. Two very swollen nuclei (arrows) in close proximity to the parasite are highlighted. Scale bar = 2.5 µm. Fig. 12. Peripheral zone of a D. phoxini-infected cerebellum in close proximity to a metacercaria. A rodlet cell (arrow) is evident amongseveral brain cells with swollen nuclei (arrowheads) and rarefied cytoplasm. Scale bar = 4.3 µm. Fig. 13a. Brain cells (arrowheads)distant from a metacercaria. Cells appear less degenerated and have only slightly swollen nuclei. Scale bar = 4.8 µm. Fig. 13b.Granule cells (arrowheads) in an uninfected brain. Brain cells possess nuclei of normal shape and size with more cytoplasm. Scalebar = 3.7 µm. Fig. 14. High magnification of the tegument of D. phoxini and a brain cell. Two secreting bodies (arrows) of para-site tegument can be seen discharging microfilamentous material (arrowheads) into the zone between the metacercaria and

brain cells of the host. A degenerated nucleus (�) is also highlighted. Scale bar = 0.8 µm

Dezfuli et al.: Metacercariae of minnow brain

and for ingestion via the mouth were likely to be low(Stein & Lumsden 1971, Erasmus 1972, Bock 1988, Wit-trock et al. 1991). The recent research of Goater et al.(2005) provided morphological evidence to suggestthat certain species of metacercariae undergo complexdevelopmental changes associated with feeding intheir intermediate host. The observation of detachedbrain tissue within the oral suckers of attached metac-ercariae in the present study would suggest that thisspecies feeds on host tissues. The work of Bibby & Rees(1971) on extracorporeal digestion in D. phoxini metac-ercariae and the finding of acid phosphatase through-out the metacercaria would suggest that intracellulardigestion is occurring and that the parasites are feed-ing. Based on the results of an experimentally infectedsample of minnows, Goater et al. (2005) suggested thatminnows are either able to tolerate a certain level ofdamage to their brains or are able to regenerate orrepair brain tissue. Post-mortem examination of min-nows in the current study revealed that the minnowshad light infections (range 1 to 38) compared withthose (1300 metacercariae) reported by Rees (1955).Therefore, the observed levels of infection and theresults from the current study correspond to the find-ings of Goater et al. (2005).

Only a few records relating to the host’s immunereaction in response to Diplostomum spp. infectionsexist (Bortz et al. 1984, Stables & Chappell 1986). Sim-ilarly, the host cellular reaction in Diplostomum-basedhistopathological studies has been largely overlooked(Rees 1955, Hoffman & Hoyme 1958, Shariff et al. 1980,Baghaei 1981). One of the main objectives of the cur-rent study was to look closely at the host–parasiteinteraction and to characterise the cells present withinthe interfacing zone. One interesting observation wasthe presence of rodlet cells within this zone, the onlyhost inflammatory cells documented in this sub-sampleof D. phoxini-infected Phoxinus phoxinus brains.Rodlet cells are, as far as we are aware, exclusive tofish and are associated with the epithelial tissues of vir-tually every fish species (Morrison & Odense 1978).Recent research by a number of workers lends supportto the theory that rodlet cells have an endogenous fishorigin (Manera & Dezfuli 2004, Giari et al. 2006, Reite& Evensen 2006, Dezfuli et al. 2007). Although theirprecise function is unknown, it is thought that rodletcells are a type of inflammatory cell akin to otherpiscine inflammatory cells (e.g. mast cells/eosinophilicgranule cells, epithelioid cells and mesothelial cells)(Dezfuli et al. 2000, Reite 2005, Reite & Evensen 2006).Most of the rodlet cells that were found in the currentstudy were observed in the epithelial lining of the ven-tricles of the optic lobes and in sites close to the tegu-ment of metacercariae. This observation fits well withLeino’s (1996) suggestion that rodlet cells react to the

presence of parasites on the epithelial surface, and thatcellular secretions are possibly antibiotic in nature.This theory is also supported by the work of others thathave investigated fish protozoan (Leino 1996, Dezfuliet al. 2004) and metazoan parasitic agents, all of whichappear to induce the recruitment of rodlet cells to thesite of infection and/or to adjacent tissues (Dezfuli et al.1998, 2000, 2003, Palenzuela et al. 1999, Bosi et al.2005, Reite 2005, Reite & Evensen 2006, the presentstudy).

While there have been numerous studies on theultrastructure of metacercariae (Stein & Lumsden1971, Strong & Cable 1972, Higgins 1980, Bock 1988,Wittrock et al. 1991), only a few have focused on that ofdiplostomatids (Bibby & Rees 1971, Mitchell 1974,Goater et al. 2005). Of these, the detailed study byBibby & Rees (1971) on the epidermis of Diplostomumphoxini metacercariae described 3 types of secretorybodies that were regularly arranged in the outer epi-dermal layer, but did not comment on the possiblefunction of the secretions. The numerous secretorybodies found in the tegument of D. phoxini in the pre-sent study were observed to release a loose, microfila-mentous material into the zone between the host andthe parasite. In addition to these bodies, many bodiesthat had already emptied their contents were also evi-dent, usually in that part of the parasite’s tegumentadjacent to a host cell (Fig. 3f). The brain cells in thisregion characteristically bore flattened nuclei andwere in a partial or complete degenerative state. How-ever, without knowing the precise chemical nature ofthe secreted product (which we are investigatingwithin our laboratory at the time of writing), we canonly hypothesise that the compound is secreted to pro-tect the metacercaria from the host’s cellular response.

Acknowledgements. We thank the technical staff of theElectron Microscopy Centre at the University of Ferrara fortheir assistance and expertise. This study was supported inpart by grants from the Italian Ministry of the University andScientific Research and Technology.

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Editorial responsibility: David Marcogliese,Montreal, Quebec, Canada

Submitted: October 12, 2006; Accepted: February 7, 2007Proofs received from author(s): March 26, 2007